The Molecular Mechanisms of Antifungal Drug Resistance In Pathogenic Fungi
Date
2016Metadata
[+] Show full item recordAbstract
Pathogenic fungi including Candida albicans, cause oral, systemic, and vaginal infections, mostly in immune-compromised individuals. Azoles are the most common antifungals used in treating these infections. The most significant mechanisms of azole resistance include alterations or overexpression of the target enzyme, and overexpression of at least three distinct efflux pumps. Recently, azole-resistant vaginal C. albicans isolates have been detected in patients with recurring and refractory vaginal infections. However, the mechanisms of resistance in vaginal C. albicans have not been studied in detail. In oral and systemic resistant isolates, over-expression of ABC transporters Cdr1p and Cdr2p and the major facilitator transporter Mdr1p is associated with resistance. This is consistent with the molecular mechanisms of drug resistance observed in this study for the vaginal C. albicans clinical isolates.
Another important mechanism of azole resistance in C. albicans is overexpression and/or point mutation of ERG11, which is a rate-limiting enzyme in the ergosterol biosynthetic pathway. Other antifungals also target the ergosterol pathway, including
fenpropimorph,(morpholine) which targets Erg24p, and terbinafine (allylamines), which targets Erg1p enzymes. Aberrant levels of ergosterol may affect many cellular processes. Recent studies have used deletion mutants of the ergosterol genes to analyze their cellular phenotypes. Some of these mutants showed severe growth defects, compromised respiration, weak cell wall, and lower tolerance to osmotic stress. However, only seven of total 25 ergosterol biosynthetic genes (ERG genes) can be deleted as they are non-essential. This thesis studies the ergosterol biosynthetic pathway in detail. All 25 S. cerevisiae ERG genes were overexpressed under a galactose inducible promoter. Nine of the 25 strains overexpressing the ERG genes showed severe growth defects. Furthermore, phenotypic changes in these strains were compared to wild-type under various stress agents. These agents affect several cellular processes that include cell wall biosynthesis, respiration, protein synthesis, osmotic stress, iron and calcium metabolism. A majority of the overexpressed strains were affected by high salt or by a calcium chelator. Two of the nine slow growing strains were affected by all the stress agents used. This project increases our understanding of the ergosterol pathway, and may identify potential targets for future drug design.
Table of Contents
The molecular mechanisms of antifungal drug resistance in pathogenic fungi -- A combination fluorescent assay demonstrates increased in efflux pump activity as a resistance mechanism in azole-resistant vaginal candida albicans -- Phenotypic changes resulting from over-expression of ergosterol biosynthetic genes in saccharomyces cerevisiae -- Altered antifungal drug susceptibilities in saccharomyces cerevisiae strains overexpressing ergosterol biosynthesis genes -- Conclusion and future direction
Degree
Ph.D. (Doctor of Philosophy)